Compositions and methods for use in analytical reactions

ABSTRACT

Compositions, methods, substrates and systems for use in analysis of single molecule reactions and particularly single molecule nucleic acid sequence analysis. Compositions that include non-reactive, distinguishable or undetectable competitive substrates for the reaction system of interest are provided, as well as their use in systems and substrates for such applications, such compounds typically preferably polyphosphate chains or analogous structures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Provisional U.S. Patent ApplicationNo. 61/065,439, filed Feb. 12, 2008, the fill disclosure of which ishereby incorporated herein by reference in its entirety for allpurposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

BACKGROUND OF THE INVENTION

Methods of determining the sequence of nucleotides in nucleic acids haveundergone substantial changes from the original adoption of gel-basedSanger sequencing, through four color capillary electrophoresis basedapproaches, both of which relied upon the electrophoretic separation ofnested synthesis fragment sets to identify sequentially terminatedsynthesis products, and as a result, identify each successive base inthe sequence. Newer approaches to sequencing rely upon “sequencing byincorporation” where each base is identified sequentially, as it isadded in a primer extension reaction. These range from pyrosequencingand other related methods that add a single base at each step and lookto see if it was incorporated, to processes that add multiple differenttypes of nucleotides each labeled with a different fluorescent dye, andidentify which base was incorporated based upon the dye incorporated atany given step. Typically, such processes require an iterative or stepby step process that employs nucleotides that include extensionterminating groups, such that after a single incorporation event, no newbases are added until the added base can be identified. The terminatinggroup is then removed and the next extension step is allowed to proceed.

In still more elegant methods, individual molecular complexes areobserved in real time, as they incorporate labeled nucleotides. Theincorporation event provides a characteristic optical signal that, alongwith a spectrally distinct dye, identifies both the incorporation eventand the type of base incorporated. In such methods, the labeling groupis often provided coupled to the phosphate chain of the nucleotideanalog beyond the alpha phosphate, resulting in cleavage of the labelfrom the nucleotide upon incorporation. This allows both the synthesisof an entirely native strand of nucleic acid, and the release of alabeling group that might otherwise confound the observation andanalysis.

The present invention provides improved compositions, methods andsystems for performing single molecule real time analyses, andparticularly single molecule, real time nucleic acid sequence analysis.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compositions, substrates, methods, andsystems that employ competitive, but otherwise unreactive substrates orinhibitors in analytical reactions to modulate the rate of reaction ofthese systems. In particular, the present invention is directed to thereal time analysis of single molecule (or single complex) reactions,which employ competitively inhibiting compositions in conjunction withthe reactants for the monitored reaction. The presence of suchcompetitors provides a mechanism for modulating the rate of themonitored reaction to provide numerous advantages. In a particularlypreferred aspect, polymerase mediated, template dependent nucleic acidsynthesis is modulated in accordance with the invention by providingwithin the reaction mixture competitive inhibitors to the polymerasebinding of incorporatable nucleoside polyphosphates, that are alsopresent in the mixture. Such competitors are characterized by theirability to competitively and reversibly associate with the polymerase,with respect to such nucleoside polyphosphates, and also their inabilityto be incorporated into the synthetic reaction.

Particularly preferred is the use of unincorporatable nucleotide analogsthat are either unlabeled, or distinctively labeled, in the analysis ofpolymerase mediated, template dependent nucleic acid synthesis andsequence characterization.

Thus, in at least one aspect, the invention provides compositions,comprising a complex comprising a nucleic acid polymerase, a templatesequence and a primer sequence complementary to at least a portion ofthe template sequence. Also included is at least a first type ofincorporatable labeled nucleotide analog, and at least a first type ofunincorporatable competitive polymerase reagent, said unincorporatablecompetitive polymerase reagent being either unlabeled or differentiallylabeled from the incorporatable labeled nucleotide analogs.

Relatedly, the invention also provides methods of determining nucleotidesequence information from a target nucleic acid sequence. The methodscomprise providing the target nucleic acid sequence in a complex with aprimer sequence complementary to at least a portion of the targetnucleic acid sequence, and a nucleic acid polymerase enzyme capable ofextending the primer sequence in a target sequence dependent manner. Thecomplex is contacted with a mixture of labeled incorporatable nucleotideanalogs and at least a first unincorporatable competitive polymerasereagent that is either unlabeled or differentially labeled from theincorporatable nucleotide analogs. Target dependent incorporation of anincorporatable nucleotide analog is then detected to identify anucleotide in the target nucleic acid sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates an exemplary single molecule real timeanalysis of nucleic acid synthesis.

FIG. 2 schematically illustrates redundant nucleotide sampling in anucleic acid synthesis reaction system.

FIG. 3 schematically illustrates nucleic acid synthesis usingunincorporatable, unlabeled competitive nucleotide substrates of theinvention.

FIG. 4 shows a schematic signal profile of a sampling based sequencedetermination process using the compositions of the invention.

FIG. 5 shows an agarose gel of template dependent, polymerase mediatednucleic acid extension products in the presence of varyingconcentrations of competitive polymerase reagents.

FIG. 6 illustrates synthesis of the unincorporatable competitivepolymerase reagent Cbz-x-5P.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention generally provides methods, compositions andsystems for improved real-time analyses and particularly nucleic acidsequence analyses. In particular, the invention provides methods andcompositions for use in single molecule analysis of a desired reaction,where the reaction rate is modulated through the presence of one or morecompetitive inhibitors of the reaction of interest. In particular, theinvention includes the use of reagents that reversibly associate withone or more components of the reaction of interest to compete with thenormal progression of that reaction, in order to modulate theprogression of that reaction. By way of example, non-reactive,non-indicative reagent surrogates are included in the reaction mixturealong with the labeled reagents themselves, in order to reduce thelikelihood of observing non-specific events that are not necessarilyassociated with the reaction that was to be observed, as well as provideother advantages to such systems. Modulation of the concentration ofthese compounds within the reaction mixture allows for the modulation ofthe rate of the reaction, providing ability to improve detection of thereaction of interest, enhance stringency of such reactions, and thelike.

In one preferred context, the invention is directed to compositions foruse in real-time nucleic acid analysis that employ competitive reagentsto nucleoside polyphosphates, in order to modulate the rate ofpolymerase mediated nucleic acid synthesis. In this context, thecompositions of the invention will typically include, in addition tolabeled nucleotide analogs, surrogate or inhibitor compounds that arecapable of reversibly associating with the polymerase enzyme in a mannerthat is competitive to the incorporatable nucleoside polyphosphatecompounds in the reaction mixture. These surrogate compounds cannot beincorporated in a primer extension reaction, and are either unlabeled,or are labeled in a fashion that allows for their ready distinction fromthose labeled nucleotides or nucleotide analogs that can beincorporated. Such surrogate compounds typically comprise a structurethat is mimetic of a nucleoside polyphosphate in its interaction withthe polymerase, but is non-incorporatable.

Thus, in one aspect, the compounds used in the invention will typicallycomprise a polyphosphate portion that is coupled to a cyclic and/oraromatic portion that mimics the nucleoside portion of a nucleotide. Insome contexts, nucleoside polyphosphates may be employed as thesurrogate compounds, but in which the structure of the compound isadjusted to render it unincorporatable, or substantiallyunincorporatable. Such compositions are described in greater detail,below. By unincorporatable is generally meant that a given compound willeither not be incorporated by a polymerase enzyme in template dependentprimer extension or incorporated at such a low level, e.g., at less than5%, preferably less than 15, and more preferably less than 0.1% of thefrequency of a corresponding nucleoside polyphosphate employed in thegiven reaction mixture, i.e., a labeled nucleoside tri, tetra, penta,hexa or heptaphosphate.

As stated above, in particularly preferred aspects, the presentinvention is directed to improved methods and compositions used inperforming single molecule real time nucleic acid sequencing byincorporation, also termed SMRT™ sequencing. As noted previously, SMRT™sequencing methods typically employ a nucleic acid synthesis complexthat includes a polymerase enzyme, e.g., a DNA polymerase, a templatesequence, and a primer sequence that is complementary to at least aportion of the template sequence. In typical primer extension reactions,the polymerase extends the primer sequence by incorporating additionalnucleotides that are complementary to the next nucleotide in theunderlying template sequence. In the real-time monitoring processes usedwith the invention, the reaction employs four distinctively labelednucleotides, e.g., each labeled with a distinguishable fluorescentlabel. The complexes are then configured such that upon incorporation ofa given base, a characteristic optical signal is produced, that bothsignals an incorporation event and allows identification of the type ofbase incorporated.

In some cases, this configuration involves the immobilization of thecomplex within an optically confined region, such that an incorporatingnucleotide is observable for a period of time that is characteristic ofthat incorporation. In particular, upon incorporation, a labelednucleotide will be retained within or proximal to the active site of theenzyme. Examples of such optically confined regions include regions ator near a surface of a transparent substrate that is illuminated usingtotal internal reflection (TIRF) spectroscopy to illuminate only speciesthat are very close to the substrate surface. In such systems,nucleotides that are being incorporated into a complex immobilizedwithin the illumination region at or near the surface, will bepreferentially illuminated, and as a result, distinguishable over other,non-incorporated molecules. Typically, the complexes are provided in aconfiguration that provides for the optical resolution of individualmolecular complexes, to permit single molecule (or single complex)elucidation of nucleic acid synthesis. Such single moleculeconfiguration may include providing complexes diluted over a surfacesuch that sufficient space is provided between the individual complexesto provide for optical resolution. Alternatively or additionally, it maycomprise immobilization of individual complexes in different confinedspaces, including, for example, optically confined regions as discussedbelow.

In other methods, the complex may be provided immobilized within anoptically confined structure, such as a zero mode waveguide (ZMW). SuchZMWs provide for an illumination region that is confined in threedimensions, as opposed to only one. In particular, a nanoscale apertureis provided through a metal cladding layer that is disposed over atransparent substrate, to define the “core” of the ZMW. This nanoscalewell structurally confines the illumination to the dimensions of thecore. Further, where the cross sectional dimensions of the core are inthe nanoscale regime of, e.g., between about 20 and about 500 nm, itwill not permit passage of light of a frequency higher than a cutofffrequency from passing through the core. Instead, light illuminating oneend of the core will be subject to evanescent decay through the core,resulting in a shallow illuminated region within the core, thusconfining the illumination in the third dimension. By immobilizing acomplex upon the transparent “floor” of the ZMW, one can selectivelyilluminate and observe interactions that occur at or around the complexwithout excessive interference from other reagents in the overallreaction mixture. The complex is then exposed to fluorescently labelednucleotide analogs that are preferably labeled upon a phosphate groupthat is released upon incorporation.

One can identify the fluorescent nucleotides that are incorporated basedupon their characteristic signal profile, which typically includes alonger retention time within the illumination region or volume ascompared to non-incorporated molecules, and free labeled polyphosphategroups. Further, based upon the spectral characteristics of thefluorescent signal, one can then identify the type of base associatedwith such incorporation events. This process is schematicallyillustrated in FIG. 1.

As shown in FIG. 1, Panel I, a polymerase/template/primer complex 102 isprovided immobilized within an illumination volume of a zero mode waveguide (ZMW) 104. Because of the dimensions of the ZMW 104, illuminationdirected at the ZMW from the bottom surface (shown as the dashed arrow106), only penetrates a short distance into the ZMW, effectivelyilluminating only a small volume therein (as shown by the dashed line108). As labeled nucleotides (shown as A, T, G and C) diffuse quickly inand out of the illumination volume, they are only transientlyilluminated, thus yielding, at best, extremely short fluorescent signalsthat are detected through the bottom of the ZMW 104, shown as briefspikes 110, in the signal traces shown in Panel II, which corresponds tothe schematic illustrations above the plots. When a nucleotide isincorporated by the polymerase into the growing nascent strand in primerextension, it is retained within the illumination volume for a periodthat exceeds transient diffusion and produces a longer fluorescentsignal 112, as a result. Because each type of nucleotide bears aspectrally distinguishable label, its incorporation can be independentlyobserved/identified (shown by the multiple traces in Panel II of FIG.1). These characteristic signal profiles are then used to identifywhether a base was incorporated and which base it was.

In still other processes, the reagents of the system are configured toprovide an optical signal primarily only in the event of incorporationby the complex. For example, such systems include fluorescent energytransfer dyes that produce signal only when in proximity to one another(donor-acceptor pairs), or sufficiently separated from one another(donor-quencher pairs). For examples a donor dye may be provided coupledto the polymerase in the complex, while the acceptor is coupled to anucleotide. Upon incorporation, the two dyes are brought into sufficientproximity to affect energy transfer and produce a characteristic signal.Conversely, one can employ a donor-quencher pair on the nucleotide,where one of the donor or quencher is provided coupled to anincorporated portion of the nucleotide, e.g., the nucleobase, while theother member of the pair is provided upon tie released phosphate groups.Upon incorporation and hydrolysis of the phosphate chain, the quencheddye diffuses sufficiently away from the quencher dye, to allow acharacteristic signal indicative of incorporation. (See, e.g., U.S. Pat.No. 6,232,075, incorporated herein by reference in its entirety for allpurposes).

These real time processes benefit from a number of advantages,including, for example, speed of base calling, increased read-lengthsfrom naturally processive polymerases operating in what is closer to anatural environment, low reagent consumption, and others.Notwithstanding these advantages, there remain areas where such systemscould still be improved. In particular, because the foregoing systemsoften rely upon the retention of the labeled nucleotide within anobservation region that results from the specific interaction of thenucleotide with a polymerase enzyme, they can be adversely impacted bynon-specific interactions in that same region that yield similarretention. Such interactions may include, for example, non-specificinteractions between nucleotides and the polymerase enzyme, such asbinding of incorrect nucleotides for the next incorporation space,surface adsorption of nucleotides on the enzyme. Alternatively oradditionally, such interactions may stem from non-specific interactionsbetween the nucleotides and other parts of the system, such as thesubstrate surfaces that lie within the observation region, also termed“sticking”.

By way of example, in the case of nucleic acid sequencing in anoptically confined region, a polymerase in the complex will randomlysample the nucleotides proximal to its active site until it finds thecorrect nucleotide to be incorporated in the primer extension reaction,i.e., that is complementary to the next base in the template sequence.Typically, this random sampling will occur much more rapidly than anincorporation event, and thus, will not provide a confounding signalevent. However, in some cases, multiple samplings of the same type ofbase without incorporation, may appear similar to an incorporationevent, and thus increase the possibility of an incorrect base call. Thisproblem can be further enhanced in reaction mixtures that includerelatively low concentrations of the nucleotides, as the ability forother, different nucleotides to compete out a repeatedly samplednucleotide will be decreased, thus increasing the likelihood of repeatedsampling.

In another example, a nucleotide that is not being incorporated, or evensampled by the polymerase in the complex, may nonetheless, becometemporarily or permanently immobilized within the observation region,and thereby become detectable for an extended period of time that canagain, provide a confounding signal, and again, a potential for anincorrect base call.

II. Competitive Substrates

The present invention addresses the issues noted above by providingcompositions that include competitive reagents to the labeled reagentsused in the reaction of interest, such as labeled nucleosidepolyphosphates used in the polymerase mediated polymerization reaction.The use of such competitive reagents not only reduces potential for thenon-specific interactions described above, but also allows for thebetter control of the timing and/or rate of reactions, e.g.,incorporation events, to suit the needs of a particular application orsystem. As used herein, the phrase “competitive polymerase reagent”refers to a compound that interacts with a polymerase or polymerizationcomplex (or component of such complex), in a competitive fashion withincorporatable nucleotide reagents, such as nucleoside polyphosphates,including for example, labeled nucleoside tetra, penta orhexaphosphates, including those that are fluorescently labeled, e.g., asdescribed in U.S. Pat. Nos. 6,936,702, and 7,041,812. For purposes ofthe invention, the competitive reagents used herein exclude naturalproducts of the reaction of interest. Thus, for example, a competitivepolymerase reagent, and particularly an unincorporatable competitivepolymerase reagent excludes the natural products of the incorporation ofa given nucleotide or nucleotide analog into a nascent nucleic acidstrand, to the extent such products may compete with the nucleotides ornucleotide analogs in association with the polymerase. For example, suchcompetitive reagents exclude released polyphosphate components thatspecifically result from nucleotide or nucleotide analog incorporationby a polymerase. Notwithstanding the foregoing, in some cases, excessamounts of such polyphosphate components may be added as the competitivereagents. Such excess amounts would typically be in line with therelative concentrations set forth herein, and in such concentrationswould fall within the scope if the invention, i.e., they would farexceed amounts of such compounds that result from nucleotide ornucleotide analog turnover.

In a particularly preferred aspect, the compositions of the inventionincorporate, in addition to the labeled nucleotide analogs, unlabeled,or differentially labeled, and unincorporatable nucleotide analogs orcompounds that mimic nucleotides or nucleotide analogs in theirinteraction with polymerase enzymes.

As noted previously, in accordance with the invention, the competitivenucleotide analogs or mimics thereof, are both unincorporatable by thepolymerase enzyme in a primer extension reaction, and are eitherunlabeled or otherwise undetectable in the analytical system, or areotherwise easily distinguished from the other detectable andincorporatable nucleotide analogs that are of real interest in theanalysis. For ease of discussion, these may be referred to hereafter as“unlabelled” nucleotide analogs. These unlabeled compounds compete withthe labeled analogs for the non-specific interactions that can yield theproblems alluded to previously.

For example, FIG. 2 schematically illustrates a system that repeatedlysamples a given incorrect nucleotide or type of nucleotide, in the caseshown, a labeled A. In particular, the labeled A is repeatedly sampled,but not incorporated, as the next added base should be a T. However,because of the multiple sampling of labeled A's, the resulting signalillustrated in an exemplary signal plat, below the schematic of thereaction, can appear more like a prolonged retention time signalassociated with an actual incorporation event, e.g., as shownsubsequently for the ultimately incorporated T, again as schematicallyillustrated in the plot beneath the illustration of the reaction.Although shown as only As and Ts within the reaction environment, itwill be appreciated that even in preferred situations where all baseswill be present, that the probability of a given, labeled base beingrepeatedly sampled by the enzyme, remains high, even if one does notaccount for repeated sampling of the identical proximal base. Inparticular, if one assumes instantaneous diffusion of a given nucleotideaway from the complex following incorrect sampling, and perfectnucleotide distribution of all nucleotides within the reaction mix, theprobability of a duplicative sampling of the same type of nucleotide inthe reaction would be 25%. As alluded to, however, it would be expectedthat some amount of repeated sampling of the identical nucleotide wouldoccur, especially where the relative concentration of other nucleotidesin the reaction mixture is low.

In accordance with the invention, however, the reaction mixture includesunlabeled and unincorporatable bases that compete for the nonspecificinteraction with the labeled bases. As such, the probability of a giventype of labeled base being repeatedly sampled will be reduced as aresult of this competition. Further, because these unlabeled nucleotideswill neither be incorporated nor detectable, they will have no impact onthe incorporation events, other than to modulate their frequency.Accordingly, one can adjust the concentration of these competitors tobest suit the desired applications, e.g., reduce redundant sampling,etc.

This is schematically illustrated in FIG. 3, which illustrates anidentical set of reaction events as shown in FIG. 2, but whereinundetectable competitive nucleotide analogs are used in conjunction withthe labeled nucleotides. In particular, the sampling of the fluorescentnucleotide analog (illustrated as an A with a resulting signal profileshown by the solid plot), is interspersed by the sampling ofundetectable nucleotide analogs (also As, and shown as a dashed line,although no signal would actually occur). Accordingly, even if thepolymerase repeatedly samples the same type of nucleotide analog, thepresence of competitive analogs will separate any signal events fromeach other and render them distinguishable from actual incorporationevents.

Although described and illustrated with reference to methods wheremultiple different types of nucleotide analogs are present at the sametime, e.g., A, G, T, C, and/or U, as well as their unincorporatablecounterpart analogs, i.e., the unincorporatable analog of the labeledand incorporatable A, G, T, C, and/or U analog, respectively, it will beappreciated that the methods of the invention are also applicable tosystems in which single nucleotide analogs are being interrogated indistinct steps, e.g., where a polymerization complex is interrogated orcontacted with only one type of nucleotide analog at a time, i.e.,bearing one type of nucleobase (adenine, guanine, thymine, cytosine,uracil, inosine and the like).

In such cases, it will be appreciated that the unincorporatablecounterpart nucleotide analog may likewise be present as the only typeof unincorporatable analog. Alternatively, in some cases, it may beadvantageous to provide a plurality of different types ofunincorporatable analogs while providing only a single type ofincorporatable analog. Conversely, there may also be situations in whichone desires to modulate sampling of only a certain type of analog. Insuch cases, while multiple different types of incorporatable analogs maybe present in the reaction mixture, only a single type ofunincorporatable analog, or less than all four types of unincorporatableanalogs, may be present in the mixture.

In addition to providing the ability to modulate the rate ofincorporation of labeled analogs, it will also be appreciated that theuse of unincorporatable analogs of the invention also provides theability to maintain elevated concentrations of labeled analogs even inthe face of improving kinetics of engineered polymerases. In particular,improvements in engineered polymerases useful in the preferredsequencing applications described herein, have resulted in substantiallyreduced Km values for the enzymes relative to the labeled analogs. As aresult, optimal reaction conditions for such enzymes result in lowerconcentrations of labeled analogs that could potentially result inreaction limiting amounts of such analogs, thus potentially reducingoverall ability to synthesize, and consequently obtain long individualmolecule read lengths of nucleic acid sequences. By providingcompetitive, unincorporatable analogs along with the incorporatableanalogs, one can effectively mediate the effects of higher analogconcentration through competition with unlabeled, unincorporatablenucleotide analogs.

In addition to the foregoing, and without being bound to any particulartheory of operation, it is also believed that processivity of thepolymerase, as well as its resistance to certain negative photoinduceddamage events, may be improved when the polymerase has bound in itsactive site a nucleotide or nucleotide analog in preparation forincorporation in an extension reaction. For example, it is believed thatfor certain polymerases, lack of a nucleotide within its active siteprovides an increased opportunity for the 3′ end of the nascent strandto transition into the exonuclease function of the polymerase, even whenexonuclease activity has been engineered out of the enzyme. In thecontext of the sequencing methods described herein, this couldpotentially lead to pauses during processive synthesis or an increasedpossibility of dissociation of the overall complex. In such cases, thepresence of the competitive unincorporatable nucleotides of theinvention provides active site coupling analogs without consequentincorporation.

In an alternative or additional configuration, the nucleotide basedcompetitive reagent compositions of the invention may be directlyemployed in identifying sequence elements, despite not beingincorporated in a nascent nucleic acid strand. In particular, Theunincorporatable nucleotide analogs of the invention, while not beingincorporatable, may be nonetheless capable of specifically associatedwith the polymerase enzyme. That is, the polymerase will sample theunincorporatable nucleotides, retaining them within the active site fora greater length of time than nucleotides that are not complementary tothe position in the template nucleic acid, and release them when theycannot be incorporated. By providing different types of nucleotide ornucleoside analogs, e.g., mimetic of A, G, T C, and/or U, bearingdistinguishable labels, e.g., spectrally resolvable fluorophores orother labeling groups, one can monitor the sampling of these nucleotidesas an indication of the nucleotide that is next to be incorporated. Forexample, one may provide labeled, unincorporatable nucleotide analogs atconcentrations in excess of incorporatable nuclotides, e.g., 2×, 5× oreven 10× or greater. Each incorporation of an incorporatable nucleotidewill, by virtue of the excess concentration, be preceded by repeatedsampling events of the unincorporatable nucleotides, which will eachcarry its associated signal event. The incorporatable nucleotides maythen either bear no label, or preferably, bear a label that isdistinguishable from the unincorporatable nucleotides, so as to mark thetermination of the sampling of a given base and proceeding onto the nextbase in the sequence. In such cases, it may be desirable to label allincorporatable nucleotides with a single type of fluorophore, i.e.,indistinguishable from the label groups on the other types ofincorporatable nuclotides present, but distinguishable from all of theunincorporatable nucleotides.

The signal detection for the foregoing process is schematicallyillustrated in FIG. 4. In particular, FIG. 4 shows a schematicillustration of a set of signal traces from a single molecule sequenceby incorporation reaction. As shown, the plot shows five signal traces.One for each type of differentially labeled unincoporatable nucleotideanalog (indicated as A′, T′, G′ and C′, as well as a trace for thesignal associated with the type of label coupled to the incorporatablenucleotide (labeled as “I”). As shown, repeated sampling of the cognateunincorporatable nucleotide analog, e.g., A′, provides an iterative setof signal events 402, followed by a signal 404 on the I trace indicatingconclusion of the incorporation event. This pattern is repeated for thenext base to be incorporated (indicated by iterative signals 406 in theT′ trace, followed again by the incorporation signal 408, in the Itrace, and again by the iterative sampling signal 410 in the A′ tracefollowed by the incorporation signal 412 in the I trace. Because theseunincorporatable nucleotides are mimetic of the base to be incorporated,they possess a longer retention time in the active site than the analogthat is not complementary to the next base in the template, and as such,provide a signal profile that is distinguishable from random, incorrectsampling, e.g., as indicated by transient signal events 414. Suchiterative sampling may include two, three, four, five, ten or greaterthan ten signal events for each incorporation.

As noted above, the competitive reagents used are going to benon-reactive in the reaction of interest. In preferred aspects, andwithout being bound to any particular theory of operation, thecompetitive compounds may possess structures similar to nucleotides orportions thereof, such that they can competitively interact with thereaction of interest, e.g., through association with the polymeraseactive site. By way of example, such structures may comprise apolyphosphate component, e.g., a pyrophosphate, triphosphate,tetraphosphate, pentaphosphate, or longer phosphate chain, so that thecompound mimics one or more of a nucleotide or the product of apolymerase mediated incorporation reaction, which is capable ofcompetitively interacting with the polymerase, relative to thenucleotide analogs.

In certain preferred cases, additional components may be coupled to thepolyphosphate component that mimic other portions of the nucleotide ornucleotide analog. By way of example, the polyphosphate component may becoupled to a cyclic and/or aromatic component that may structurallymimic the nucleoside component in its interaction with the polymerase.Such structures are generally illustrated by the following structure:

P—(P)_(n)-A;

where P is a phosphate or phosphonate group, n is an integer from 1 to6, and A includes a cycloalkyl or aryl group, a carbohydrate group, orthe like.

In the case of nucleotide analogs used in analytical primer extensionreactions, e.g., in nucleic acid sequence analysis, such nucleotideanalogs will be unincorporatable in such primer extension reaction bythe polymerase used. Further, in preferred aspects, suchunincorporatable analogs will typically still be capable of interactionwith the polymerase, e.g., active site binding, but will be unable to beincorporated in a primer extension reaction. In preferred aspects, thisis accomplished by providing nucleotide analogs that possessunhydrolyzable groups within the phosphate chain, such that thephosphoester linkage between the analog and the primer strand, cannot beformed, as mediated by the polymerase. One particularly effectiveapproach to producing an unincorporatable nucleotide analog includesreplacing the phosphoester linkage between the alpha and beta phosphateof a nucleoside polyphosphate with a nonhydrolyzable linkage.

One example of such an analog is illustrated below, where tie oxygengroup between the alpha and beta phosphate groups is replaced with anunhydrolyzable linkage, such as the illustrated amino group.

Although illustrated as an amino linkage, it will be appreciated that avariety of other linkages may be used between the alpha and betaphosphates, e.g., an amino, methyl, thio, or other linkages nothydrolyzed by polymerase activity. Additionally, although illustrated asincluding three phosphate groups analogous to a nucleoside triphosphate,it will be appreciated that other polyphosphate configurations may beemployed in the invention, including, for example, tetraphosphateanalogs, pentaphosphate analogs, hexaphosphate analogs, and the like.

Thus, the structures employed in certain preferred aspects of theinvention may generally be described with reference to the followingstructure:

where R₁ comprises a linking group that is non-hydrolyzable by thepolymerase enzyme being used. Particularly preferred linkages includeamino linkages, alkyl linkages, e.g., methyl, and thio linkages. WhileR₂ may comprise oxygen, in some preferred aspects, it will includeadditional phosphate groups, e.g., mono-, di-, or triphosphate groupscoupled to the gamma phosphate group. Alternatively or additionally, theR₂ group may include, in addition to or instead of additional phosphategroups, labeling functionalities that provide for the detection of thecompetitive substrates, but still permit its distinguishing from theincorporatable nucleotides. In other aspects, the R₂ group (orcorresponding groups on other structures described herein, i.e., groupR₉ discussed with reference to other compounds, below), may includemoieties that provide other functionalities to the reaction system otherthan as a labeling group. For example, R₂ may comprise an agent thatreduces the potential for photodamaging effects on a polymerase enzyme,either coupled directly to the terminal phosphate group, or through alinking group.

Such moieties include, for example, triplet state quencher moietiesthat, when bound in the active site of the polymerase, may function toreduce the level of triplet state fluorophores within or near the activesite of the enzyme. A variety of reducing agents or anti-fade agents maybe used as triplet state quenchers, including without limitationascorbic acid, dithiothreitol (DTT), mercaptoethylamine (MEA),β-mercaptoethanol (BME), n-propyl gallate, p-phenylenediamene (PPD),hydroquinone, sodium azide (NaN₃), diazobicyclooctane (DABCO),cyclooctatetraene (COT), as well as commercially available anti fadeagents, such as Fluoroguard (available from BioRad Laboratories, Inc.,Hercules, Calif.), Citifluor antifadants (Citifluor, Ltd., London, UK),ProLong, SlowFade, SlowFade Light (Invitrogen/Molecular Probes, Eugene,Oreg.), and 3-nitrobenzoic acid (NBA). As will be appreciated, in thecontext of the invention, the foregoing agents may optionally oradditionally be included separately from the dye labeled compounds,e.g., as reaction mixture additives. Alternatively or additionally,oxygen scavenging groups may be provided to remove radical oxygenspecies in or around the enzyme. Examples of oxygen scavengers include,for example, lycopene, α, β, and γ-carotene and their analogs,antheraxanthin, astaxanthin, canthaxanthin, (See, e.g., Carcinogenesisvol. 18 no. 1 pp. 89-92, 1997), neurosporene, rhodopin, bixin, norbixin,zeaxanthin, lutein, bilirubin, biliverdin, and tocopherols (See, e.g.,Biochem Soc Trans. 1990 Dec; 18(6): 1054-6 ref.) as well as polyenedialdehydes (Carcinogenesis vol. 18 no. 1 pp. 89-92, 1997) melatonin,vitamins E (α-tocopheryl succinate and its analogs) and B₆ (pyridoxine1and its derivatives). Other chemical oxygen scavengers are alsoavailable, e.g., hydrazine (N₂H₄), sodium sulfite (Na₂SO₃),hydroxylamine, glutathione, and N-acetylcysteine, histidine, tryptophan,and the like. In addition to the foregoing, in many cases, the amount ofsinglet oxygen quenchers or scavengers may be reduced or eliminated byphysically excluding oxygen from the reaction of interest by, e.g.,degassing reagents, perfusion with inert gases, or the like. In additionto the foregoing, as an additional or alternative to the foregoingcompounds, anti-oxidants may also be provided in the reaction mixture,including, e.g., Trolox and its analogs U-78715F and WIN62079, a solubleform of vitamin E, having a carboxyl substitution, or in the case ofanalogs, other substitutions, in place of the vitamin E phytyl sidechain, ascorbic acid (or ascorbate), butylated hydroxytoluene (BTH), andthe like.

Use of such triplet state quenchers or oxygen scavengers as a functionalmoiety of a nucleotide analogs has been previously described inProvisional U.S. patent application Ser. No. 61/026,992, filed Feb. 7,2008, and incorporated herein by reference in its entirety for allpurposes.

The remaining substituents, e.g., R₃-R₇ are independently selected fromgroups that are known in the art to be incorporatable at these positionsin nucleotide analogs for various applications. For example, R₃-R₅ maygenerally be independently selected from O, BH₃, and S. In addition,while R₆ and R₇ are preferably H and OH, respectively, it will beappreciated that for different applications, they may each beindependently selected from H and OH.

Typically, except for structural alterations used to render themunincorporatable, and a missing or distinguishable label, thecompetitive substrates of the invention will often mirror the structureof the nucleotide analogs with which they are intended to compete. Forexample, typically all four standard nucleobases will be representedamong the competitive substrates within the reaction mixture, i.e.,Adenine, Guanine, Thymine, Cytosine, and/or Uracil, at the same orsimilar ratios to each other, as for the incorporatable nucleotideanalogs. Further, as noted above, the competitive substrates willpreferably lack any labeling groups, such as fluorescent dyes, or thelike, in order to avoid any contribution of such labels to signal noiselevels within the reaction system. However, in the event that labelingis desired to monitor the interaction between the reaction complex andthe competitive substrates, one would typically employ a labeling groupthat is distinguishable from all of the labels employed on theincorporatable nucleotides. In particular, one may employ fluorescentlabels having distinct excitation or emission spectra, so as to permittheir differential detection through either differential illumination ordifferential signal direction.

In other aspects, non-nucleotide compounds may be employed as thecompetitive reagents to the incorporatable nucleotides. Examples of suchnon-nucleotide or other competitive reagents include compounds that,with respect to binding within the active site of a polymerase, aremimetics of nucleotides or nucleotide analogs. Such compounds willtypically comprise pyrophosphate or polyphosphate compounds. Thesepyrophosphate and/or polyphosphate compounds are typically capable ofbinding to the catalytic center of the polymerase mimicking thepolymerization reaction product and thus competing with labelednucleotide analogs in the active site binding of such nucleotides. Asthese compounds are not nucleotides, they would not yield any consequentincorporation event.

The polyphosphate compounds of the invention will typically comprise oneof the following structures:

R₈(—P)_(n)—P—R₉

R₈(—P)_(n)—P

where P is selected from a substituted or unsubstituted phosphate orphosphonate group, where such phosphate groups may be joined byphosphodiester linkages, amine groups, sulfur groups, alkyl groups, orthe like as discussed elsewhere herein, R₈ comprises a substituted orunsubstituted cycloalkyl or aryl group, including, e.g., heterocyclic,bicycloalkyl, and carbohydrate groups, such as ribosyl or glucosylgroups, which are optionally coupled through alkyl linker groups, andR₉, when present, may include a detectable labeling group, e.g., anoptical or electrochemically detectable label group, such as afluorophore or fluorescent or luminescent compound or particle; and n isan integer from 1 to 6, and further provided that such compounds are notincorporatable by a nucleic acid polymerase into a nascent nucleic acidstrand.

One particularly exemplary compound of the foregoing structure includesa cyclobenzyl group linked to a pentaphosphate compound (also referredto herein as Cbz-x-5P) of the structure:

As noted previously, fewer or more phosphates may be included within thephosphate chain portion of the compound. Furthermore, it should be notedthat although this substrate is not incorporated, it may function as asubstrate in a base excision. However, this activity is negligiblecompared to sequencing/base incorporation and therefore does notinterfere with its use in the invention.

The relative concentration of the competitive substrates to theincorporatable substrates, within a reaction mixture may generally bevaried in accordance with a desired application. In particular, becausethe concentration of the competitive substrates affects the interactionsof the complex with the incorporatable nucleotides, one can modulatethose interactions by altering the ratios between incorporatablenucleotides and competitive substrates. In typical applications,however, the relative molar concentration of competitive substrate willrange from about 0.5× to about 10×, 20× or greater of the concentrationof the actual substrates (or incorporatable nucleotide analogs). Thus,the concentration ratio of unincorporatable nucleotide analogs toincorporatable nucleotide analogs will typically range from a lowerratio of from about 0.1:1, 0.2:1 0.5:1 and 1:1, to an upper ratio ofabout 2:1, 3:1, 5:1, 10:1 or even 20:1, with each iteration of theforegoing being encompassed in the disclosure hereof.

EXAMPLES

Polymerase mediated primer extension reactions were carried out invarying concentrations of a nucleotide mimetic compound to measure thecompetitive impact of such compounds on nucleotide incorporation bypolymerases. Nucleotide incorporation was measured based upon theelongation rate of the polymerization reaction in the presence ofvarying concentrations of the competitive compound, as determined fromthe change in synthesis product size, by agarose gel electrophoresis.

A DNA primer extension reaction was carried out using a short circulartemplate sequence using an exonuclease deficient modified phi29 DNApolymerase in the presence of 10 μM of dTTP, 10 μM dCTP, 5 μM AlexaFluor® 660 labeled deoxyadenosine hexaphosphate (dA6P), and 5 μM AlexaFluor® labeled deoxyguonosine hexaphosphate (dG6P), where thefluorescent label was coupled to the terminal phosphate. The reactionbuffer was 50 mM ACES at pH 7.1, with 75 mM potassium acetate and 1.5 mMMnCl₂. Different reactions were carried out in the absence of Cbz-x-5P(lane 1), or in the presence of 60 μM (lane 2), 125 μM (lane 3) and 250μM Cbz-x-5P (lane 4). A molecular weight standard was also run (shown inlane 5).

The extension reaction products were then separated on an agarose geland are shown in FIG. 5. As can be seen, increased concentration of thecompetitive compound yields a reduction in the size of the extensionproduct illustrating competitive inhibition of the overall extensionreaction and slowing of the overall extension rate of the polymerase.

Synthesis of Z-6-aminohexylpentaphosphate (Cbz-X-5P) was prepared fromcommercial 6-(Z-amino)-1-hexanol (Fluka) in a multi-step synthesis. Inthe first step, 6-(Z-amino)-1-hexanol was converted toZ-6-aminohexylphosphate using phosphorous oxychloride and aqueouswork-up. The monophosphate was activated with CDI in anhydrous DMF, theexcess of CDI was decomposed with methanol, and the resultingintermediate was treated with commercial tributylammonium pyrophosphate(Sigma) to yield Z-6-aminohexyltriphosphate (Cbz-X-3P). In a similarprocedure (CDI, methanol, pyrophosphate), the triphosphate was convertedto the final Z-6-aminohexylpentaphosphate (Cbz-X-5P). The product waspurified by reverse phase HPLC followed by ion-exchange chromatography.This synthetic scheme is further illustrated in FIG. 6.

Although described in some detail for purposes of illustration, it willbe readily appreciated that a number of variations known or appreciatedby those of skill in the art may be practiced within the scope ofpresent invention. All terms used herein are intended to have theirordinary meaning unless an alternative definition is expressly providedor is clear from the context used therein. To the extent any definitionis expressly stated in a patent or publication that is incorporatedherein by reference, such definition is expressly disclaimed to theextent that it is in conflict with the ordinary meaning of such terms,unless such definition is specifically and expressly incorporatedherein, or it is clear from the context that such definition wasintended herein. Unless otherwise clear from the context or expresslystated, any concentration values provided herein are generally given interms of admixture values or percentages without regard to anyconversion that occurs upon or following addition of the particularcomponent of the mixture. To the extent not already expresslyincorporated herein, all published references and patent documentsreferred to in this disclosure are incorporated herein by reference intheir entirety for all purposes.

1. A composition, comprising: a complex comprising a nucleic acidpolymerase, a template sequence and a primer sequence complementary toat least a portion of the template sequence; at least a first type ofincorporatable labeled nucleotide analog; and at least a first type ofunincorporatable competitive polymerase reagent, said unincorporatablecompetitive polymerase reagent being either unlabeled or differentiallylabeled from the incorporatable labeled nucleotide analogs.
 2. Thecomposition of claim 1, wherein the unincorporatable competitivepolymerase reagent comprises a polyphosphate compound.
 3. Thecomposition of claim 1, wherein the competitive polymerase reagentcomprises an unincorporatable nucleotide analog.
 4. The composition ofclaim 1, further comprising a plurality of types of incorporatablelabeled nucleotides analogs.
 5. The composition of claim 4, furthercomprising a plurality of types of unincorporatable nucleotide analogs.6. The composition of claim 3, wherein the unincorporatable nucleotideanalogs comprise a link between an alpha and beta phosphate groups thatis unhydrolyzable by the polymerase enzyme.
 7. The composition of claim6, wherein the link between the alpha and beta phosphate groups isselected from amino, thio, or alkyl.
 8. The composition of claim 1,wherein the unincorporatable competitive polymerase reagent is presentat a concentration ratio to the labeled incorporatable nucleotideanalogs of from about 0.1:1 to about 20:1.
 9. The composition of claim3, wherein the unincorporatable nucleotide analogs are unlabeled. 10.The composition of claim 1, wherein the complex is immobilized upon asolid support.
 11. The composition of claim 10, wherein the solidsupport comprises a transparent substrate.
 12. The composition of claim10, wherein the complex is immobilized upon a solid support such thatthe complex is individually optically resolvable.
 13. The composition ofclaim 10, wherein the complex is immobilized within an opticallyconfined structure.
 14. The composition of claim 1, wherein theunincorporatable competitive polymerase reagent further comprises atriplet state quencher moiety coupled to the competitive polymerasereagent.
 15. The composition of claim 2, wherein the unincorporatablecompetitive polymerase reagent comprises a group coupled to thepolyphosphate group, selected from a cycloalkyl group, an aryl group,and a carbohydrate.
 16. A method of determining nucleotide sequenceinformation from a target nucleic acid sequence, comprising: providingthe target nucleic acid sequence in a complex with a primer sequencecomplementary to at least a portion of the target nucleic acid sequence,and a nucleic acid polymerase enzyme capable of extending the primersequence in a target sequence dependent manner; contacting the complexwith a mixture of labeled incorporatable nucleotide analogs and at leasta first unincorporatable competitive polymerase reagent that is eitherunlabeled or differentially labeled from the incorporatable nucleotideanalogs; and detecting target dependent incorporation of anincorporatable nucleotide analog to identify a nucleotide in the targetnucleic acid sequence.
 17. The method of claim 16, wherein theunincorporatable competitive polymerase reagent comprises apolyphosphate chain.
 18. The method of claim 17, wherein thepolyphosphate chain comprises from 2 to 7 phosphate groups
 19. Themethod of claim 16, wherein the unincorporatable competitive polymerasereagent comprises an unincorporatable nucleotide analog.
 20. The methodof claim 16, wherein the mixture of labeled incorporatable nucleotideanalogs comprises a plurality of different types of labeledincorporatable nucleotide analogs and the at least one unincorporatablecompetitive polymerase reagent comprises a plurality of different typesof unincorporatable nucleotide analogs.
 21. The method of claim 16.wherein a ratio of labeled incorporatable nucleotide analogs tounincorporatable competitive polymerase reagent in the mixture is fromabout 0.1:1 to about 20:1.
 22. The method of claim 16, comprisingproviding the complex in an individually optically resolvableconfiguration, and optically detecting incorporation of a labelednucleotide analog in a primer extension reaction by the polymeraseenzyme in the complex.
 23. The method of claim 16, wherein theunincorporatable competitive polymerase reagent comprises an aryl orcycloalkyl group linked to a polyphosphate group.
 24. A method ofdetermining a sequence of a template nucleic acid, comprising: providingthe target nucleic acid sequence in a complex with a primer sequencecomplementary to at least a portion of the target nucleic acid sequence,and a nucleic acid polymerase enzyme capable of extending the primersequence in a target sequence dependent manner; contacting the complexwith a mixture of labeled incorporatable nucleotide analogs and at leasta first unincorporatable competitive polymerase reagent that isdifferentially labeled from the incorporatable nucleotide analogs;detecting iterative sampling of the first unincorporatable nucleotide bythe complex; and identifying a base in the template nucleic acid basedupon an identity of the first unincorporatable nucleotide analog. 25.The method of claim 24, wherein the mixture comprises a plurality oftypes of incorporatable nucleotide analogs bearing a first detectablelabel, and a plurality of types of unincorporatable nucleotide analogs,each type of unincorporatable nucleotide analog bearing a label that isdistinguishable from each other type of labeled unincorporatablenucleotide analog and the first detectable label on the incorporatablenucleotide analogs.